The current art of measuring antimicrobial efficacy does not take into account concurrent bactericidal and bacteriostatic activity. It also lacks a unifying structure for the integration of observed responses of a bacterial population to the action of an antimicrobial agent. As a result, test methods are currently based on the nature of the observation unique to the particular test, viz. the absence of visible colonies or turbidity, rather than on independent criteria of efficacy which the test would seek to measure. The relationship between test measurements and the purpose of the test are therefore elusive; even those highly skilled in the art are unable to select test procedures and to define associated protocols which can reliably relate test measurements to therapeutic efficacy criteria. Thus, the prior art, while rich in knowledge concerning effects at the level of the individual cell, suffers from the fundamental deficiency of lacking a practical, theoretical basis for relating the response of a bacterial population to the bactericidal and bacteriostatic actions of an antimicrobial agent.
Inadequacies of the prior art as it applies to the testing of antimicrobial agents in clinical practice and new drug development are summarized below.
The most common measurement characterizing the effect of an antimicrobial agent, and providing a basis for assessing its potential efficacy to combat pathogens, is the Minimum Inhibitory Concentration (MIC): it is the concentration of the agent--generally in a series of twofold dilutions--which leads to suppression of growth as observed in the test. By currently accepted standards as published in the following publications: (1) NCCLS (National Committee for Clinical Laboratory Standards) (1990a). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria--Second Edition; Approved Standard M11-A2. NCCLS, Villanova Pa.; NCCLS (National Committee for Clinical Laboratory Standards) (1990b) and (2) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically--Second Edition; Approved Standard M7-A2. NCCLS, Villanova Pa., the MIC cannot be defined as an absolute quantity and a wide tolerance of +/- one twofold dilution--a range of 300 percent--is accepted as the practically achievable accuracy of its measurement. Observation of growth transition features--from contiguous growth of colonies to their complete absence on agar media, or partial turbidity in broth dilution tests--are neglected, or subject to diverse interpretations, because their source and relevance are not understood (they also contribute to the wide variation of test results). It is therefore not surprising that the specification of MIC breakpoint values, used to judge whether a bacterial isolate will be susceptible or resistant to treatment by the tested antimicrobial agent, is subject to considerable uncertainty. Indeed, the Working Party of the British Society for Antimicrobial Chemotherapy states in the introduction to their Guide to Sensitivity Testing that "the only clear property of breakpoint antibiotic concentrations is that they are largely arbitrary" (Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1991).
In a presentation to a symposium of the American Society of Microbiology in Philadelphia, Pa., November, 1991, Samuel Schalkowsky defined the MIC as a discrete quantity on a continuous scale of concentrations of the antimicrobial agent, referred to as the Discrete MIC (DMIC). See Schalkowsky, S. (1991) Measures of Susceptibility from a Spiral Gradient of Drug Concentrations. In Antimicrobial Susceptibility Testing: Critical Issues of the 90's (Poupard et al. Ed.) pp 107-120, Plenum Press, New York 1994. Specifically, the DMIC represents the concentration of the antimicrobial agent resulting in essentially no change from the initial number of viable cells in the test population with increasing time of exposure to the drug. Reductions in the viable population will be exhibited at higher concentrations while increasing populations will result at concentrations lower than the DMIC. Schalkowsky proposed that the DMIC be measured by means of time-kill curves at concentrations near the DMIC, by obtaining their slopes for short exposure intervals, performing a regression of these slopes against their corresponding drug concentrations, and obtaining the DMIC value as the concentration at which the regression line intercepts the concentration axis, i.e. when the slope is equal to zero. Thus, the DMIC was defined by the horizontal time-exposure axis of conventional time-kill curves.
The need to assess the bactericidal activity of antimicrobial agents is well recognized. The most common estimator of bactericidal activity is the Minimal Bactericidal Concentration (MBC). It is the twofold dilution of the antimicrobial agent which reduces the original viable bacterial population by at least a factor of 1,000 (99.9%) after exposure for a (arbitrarily) preselected period of time, e.g. 24 hours. Current practice and problems associated with the MBC are documented by a subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) charged with the formulation of standardized laboratory methods for the testing of bactericidal activity. In September 1992 the NCCLS published Document M26-T as a Tentative Guideline of "Methods for Determining Bactericidal Activity of Antimicrobial Agents". This document (M26-T) follows the earlier issuance in 1986 of the Proposed Guideline, M26-P. See NCCLS (National Committee for Clinical Laboratory Standards) (1986). Methods for Determining Bactericidal Activity of Antimicrobial Agents; Proposed Guideline M26-P. NCCLS, Villanova Pa. The Tentative Guideline incorporates modifications resulting from comments received in the intervening six years in response to the Proposed Guideline, as a part of the consensus development process. The NCCLS document M26-T is therefore a highly authoritative representation of the current art of bactericidal activity testing and it clearly states that "The determination of the MBC . . . is so subject to methodologic variables that the clinical relevance of MBCs is nearly impossible to assess." Thus, again, in the prior art the relationship between test measurements and the objective of assessing the practical relevance of the test is elusive, at best.
NCCLS M26-T identifies specific problems causing uncertainty in the interpretation of bactericidal activity measurement. The four listed "biological factors" are (1) persisters, (2) paradoxical effect, (3) tolerance and (4) phenotypic resistance.
The expressing of the rate of population change on a per drug-free generation has been used by Tuomanen et al. to report results of laboratory tests dealing with the "rate of killing" of the tested antimicrobial agent. See Tuomanen, E., Cozens, R., Tosch, W., Zak, O. & Thomasz, A. (1986); the rate of killing of Escherichia coli by .beta.-lactam antibiotics is strictly proportional to the rate of bacterial growth. Journal of General Microbiology 132, 1297-1304. This Tuomanen et al. reference is cited and its conclusion paraphrased in the NCCLS M26-T document as part of the attempt to describe the above persisters factor. It did not serve to define a useful measurement of antimicrobial efficacy.
In the prior art, the MIC is frequently referred to as a measure of bacteriostatic activity. But this is a convention rather than a scientifically established fact (it has likely evolved as a result of the existence of the separate MBC measurement which refers to bactericidal activity).
In the current art assessment of the efficacy of drug combinations are made using the imprecise, twofold MIC techniques, seeking to determine the elusive "synergistic" property of a combination, i.e. whether 1 plus 1 equals 3.